The relationship of T cell receptor assembly, expression, and signaling to T cell development in the thymus has been elucidated by a variety of in vitro and in vivo experiments. We have demonstrated that the major route of TCR assembly in immature thymocytes is the association and subsequent disulfide bonding of two trimeric complexes: one consisting of TCRalpha, CD3gamma, and CD3epsilons proteins; and the other consisting of TCRbeta,CD3delta, and CD3 epsilon proteins. However, we discovered that in immature thymocytes assembly of complete TCR complexes was not necessary for surface expression as immature thymocytes expressed on their cell surfaces partial receptor complexes, referred to as clonotype- independent complexes, that consisted of CD3 components associated with a surface form of the molecular chaperon calnexin and that were able to transduce signals regulating early T cell development. Interestingly, we found that retention of nascent proteins within the endoplasmic reticulum (ER) was generally inefficient in immature thymocytes and in that a variety of different molecular chaperones are expressed on the surface of immature thymocytes that normally are completely retained within the ER. With regard to repertoire selection events in the thymus, we found a marked asymmetry in the signals required for CD4 versus CD8 commitment, in that CD8-commitment was strictly dependent upon MHC class I instructional signals whereas CD4-commitment appeared to occur by default. We developed an assay, referred to as the coreceptor reexpression assay, that was capable of detecting lineage commitment at a molecular level immediately upon termination of either CD4 or CD8 synthesis. Interestingly, we identified a specific subpopulation of TCRhiBcl2hi immature thymocytes as the earliest cells to have undergone lineage commitment. Significantly, we have developed an in vitro system in which immature DP thymocytes can be signaled to differentiated into CD4+ T cells in the absence of thymic epithelium. This in vitro system has the potential to identify receptor-ligand interactions between immature thymocytes and thymic epithelium that generate intracellular signals resulting in positive selection.